The present invention relates to a novel acyl coenzyme A (acyl-CoA) synthetase and thermophilic Pseudomonas UKSW-3733 strain which is capable of producing said acyl-CoA synthetase.
Acyl-CoA synthetase (hereinafter referred to as ACS) is an enzyme which activates a fatty acid into its CoA derivative (acyl-CoA) in the presence of coenzyme A (hereinafter referred to as CoA) and adenosine triphosphate (hereinafter referred to as ATP). This reaction can be represented as follows: ##STR1## (in which R.COOH represents a fatty acid; RCOCoA represents a CoA derivative of a fatty acid (acyl-CoA); and PPi represents pyrophosphoric acid.)
It is important from medical point of view to measure the amount of free fatty acids contained in blood since it increases to a considerable degree in the case, e.g., of diabetes. The measurement is therefore recognized as an important item in clinical diagnostic chemistry. Although various chemical methods have already been known, enzymatic methods have been generally employed for the measurement in recent years because of their accuracy and convenience. In enzymatic methods, ACS is allowed to react with free fatty acids to generate acyl-CoA, AMP, PPi, etc. in accordance with the reaction formula (1) described above, and the thus formed acyl-CoA, AMP, PPi, etc., are determined by use of other enzymes. Because of their accuracy and convenience, such enzymatic methods are now mainly used instead of the chemical methods in the measurement of free fatty acid. In search of sources, in nature, of ACS to be used in the first stage of the enzymatic methods, extensive investigations have been made, and it has been found that ACS is produced by the liver microsome of rat and by various microorganisms. Some of known ACS isolated from microorganisms are already on the market (as described, e.g., Biochemical Catalogue of Toyobo Co., Ltd. (November, 1985) and Enzyme Catalogue of Toyo Jozo Co., Ltd. (June, 1982)). In particular, the distribution of microorganisms capable of producing ACS has been extensively researched, since it is possible to culture microorganisms in quantities larger than in the case of animal cells and, hence, they can be a less expensive source of the enzyme. As a result, ACS has been discovered in various microorganisms, including, e.g., bacteria, such as Escherichia coli (European Journal of Biochem., Vol. 12, pp. 576-582, 1970), Bacillus megaterium strain M (Biochemistry, Vol. 4, pp. 85-95, 1965), and Pseudomonas 22 (Journal of Bacteriology, Vol. 105, pp. 1216-1218, 1971); yeasts, such as Torulopsis Y-8 (Joural of Bacteriology, Vol. 104, pp. 1397-1398, 1970); and actinomycetes, such as Norcadia asteroides (Journal of Bacteriology, Vol. 114, pp. 249-256, 1973). In Japanese Patent Publication No. 46757/81 is described a method for producing a highly active acyl-CoA synthetase from various microorganisms, including bacteria, yeasts, and molds, for example, Pseudomonas aeruginosa (IFO-3919), Pseudomonas synxantha (IFO-3906), Pseudomonas schuylkilliensis (IFO-12055), Candida lipolytica (IFO-0717), Gibberella fujikuroi (IFO-6604), Fusarium oxysporum (IFO-5942), Seratia marcescens (IFO-3054), and Aeromonas hydrophila (IFO-3820).
On the other hand, thermophilic bacteria belonging to the genus Pseudomonas that are incapable of producing ACS are also known Pseudomonas thermoamylolyticus (Japanese Patent Application (OPI) No. 106786/76), Pseudomonas thermophila K-2 (Izv. Akad. Nauk. SSSR, Ser. Biol., Vol. 2, pp. 271-283, 1982), and Pseudomonas hydrogenothermophila TH-1 (Agr. Biol. Chem., Vol. 41, pp. 685-690, 1977). The first is a thermophilic Pseudomonas bacterium capable of producing amylase and having an optimum temperature for growth in the range of from 60.degree. to 65.degree. C. The second and the third are hydrogen bacteria capable of growth on carbonate as the sole carbon source but having an optimum temperature for growth at around 50.degree. C.
The above-described microorganisms which grow at ordinary temperatures (e.g., 20.degree. to 37.degree. C.) produce ACSs having only a poor storage stability, and such ACSs are liable to deactivation during purification. In addition, when such an ACS is used in a composition for measuring free fatty acids, the resulting composition may become unstable within one day because of inactivation of the ACS, or inaccurate measurement results may result because of impurities contained therein. The microorganisms are also disadvantageous in that their fermentation must be carried out at ordinary temperature, and, hence, are liable to be contaminated with various germs. This can be a serious bottleneck in large scale production of ACS.
On the other hand, none of the above-described Pseudomonas bacteria having their optimum temperature for growth in the range of from 60.degree. to 65.degree. C. is capable of producing ACS. In addition, it is known that enzymes obtainable from highly thermophilic bacteria having an extremely high optimum temperature for growth (65.degree. to 80.degree. C.) are generally impractical in spite of the fact that they have excellent storage stability. This is because their optimum reaction temperature is high, and, hence, their activity markedly decreases at temperatures of from 30.degree. to 37.degree. C., at which clinical diagnosis is typically performed. Upon fermentation of the above-described Pseudomonas bacteria having their optimum temperature for growth at around 50.degree. C., hydrogen, oxygen, and carbon dioxide must be supplied to the medium at a ratio of 7/1/1 in order to maintain their growth, as they are hydrogen bacteria utilizing carbonate as the only source for their growth. Commercial fermentation of such bacteria is difficult since it cannot be controlled easily, and tends to be accompanied by the danger of explosion because of the use of hydrogen. In addition, none of the hydrogen bacteria have been known to have the ability of producing ACS.